The objective of this proposal is to develop in vitro models of the HCV replication cycle which mimics the processes of infectivity of HCV replication in patients. These in vitro systems will provide valuable tools for studying the mechanisms of HCV pathogenicity is human primary hepatocytes and facilitate the development of novel therapeutic agents. An HCV hepatocyte primary culture system using cells isolated from normal human adult and fetal liver will be developed. Because availability of these tissues on a regular basis is limited, isolated liver cells that have been infected in vitro or are yet uninfected will be frozen under controlled conditions and stored i liquid nitrogen until needed for either in vitro or in vivo (SCID) mice animal model) investigation. Human hepatocyte primary cultures obtained (either from freshly isolated or cryopreserved cells prior to HCV exposure will be characterized, according to morphology, synthesis and secretion of specific liver proteins, lactate dehydrogenase activity and specific metabolic functions. Conditions leading to long-term in vitro culture of these cells including stable cellular functions will be defined. Hepatocytes will be infected in vitro with HCV obtained from a chronically active HCV carrier, and HCV replication cycle will be characterized in detail with measurement of the level of infection by specific strand reverse transcription-polymerase chain reactive (RT-PCR). Hepatic cellular functions possibly affected by HCV will be assessed. Classes of compounds which are potential candidates for inhibiting HCV replication with a primary emphasis on RNA-dependent RNA-polymerase (RDRP) as the antiviral target site will be studied. The use of a surrogate virus replicon RNA infecting HepG2 cells and possibly primary hepatocytes with monitoring of genome replication by luciferase production will provide the required technology for large scale evaluation of possible candidates inhibiting that RDRP target site. Confirmatory testing will subsequently be performed in the HCV-human hepatocyte culture system. These cell-based assays will be complemented by biochemical enzymatic studies for elucidating detailed molecular characteristics of the newly discovered active entities against HCV replication. Following selectivity studies, novel entities will be evaluated in the animal models. In summary, a rationale and intertwined progression will be conducted by the UAB Hepatitis C Research Group in close collaboration with Emory investigators toward the discovery of novel therapeutic agents for the treatment of hepatitis C infections.